The Section on Steroid Regulation (SSR) investigates molecular mechanisms and biologic implications of modifying substances by sulfonation, a fundamental process in the biotransformation of endobiotics as well as drugs and xenobiotics. Sulfonation, the transfer of an SO3-1 group from the universal sulfonate donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor compound, is essential for normal growth and development as well as maintenance of the internal milieu. The SSR investigates enzymes that transfer the sulfonate moiety, i.e. sulfotransferases as well as the bifunctional synthetases producing the sulfonate donor. There are two classes of sulfotransfersases: one class is tightly associated with membranes, especially the Golgi complex, while the other class is composed of a super family of soluble or cytosolic enzymes (designated SULT). The former class sulfonates macromolecules such as glycosaminoglycans and proteoglycans, whereas the SULT class sulfonates low molecular weight compounds, such as hormones and neurotransmitters of which the SSR is currently focusing on the SULT2 family that sulfonatees steroids and sterols. Importantly, by modulating availability of biologically active substances, sulfonation can influence biologic activity regardless of whether compounds act in their unconjugated or sulfoconjugated form or whether they act via a genomic or nongenomic mechanism. Thus, sulfotransferases play an essential role in specific physiologic systems and their associated disorders. The gene for human SULT2B1, as a result of an alternative exon 1 and differential splicing, encodes for two mRNAs, i.e. SULT2B1a and SULT2B1. The use of exon 1A produces SULT2B1a, whereas to produce SULT2B1b exon 1B plus a portion of exon 1A is required. While SULT2B1a avidly sulfonates the steroid pregnenolone, SULT2B1b is the physiologic cholesterol sulfotransferase. While SULT2B1b is selectively expressed in a tissue-specific manner e.g. skin, SULT2B1a is essentially globally silenced. DNA analysis revealed that the proximal promoter regions of both SULT2B1 isoforms contain multiple CpG dinucleotides where cytosines are subject to methylation. SULT2B1a and SULT2B1b promoters in human cells that do not express these isoforms, are hypermethylated. In contrast, the proximal promoter of SULT2B1b in keratinocytes that do highly express this isoform, is completely unmethylated. Removal of the methyl groups leads to a striking induction of expression, whereas in vitro methylation of SULT2B1a and SULT2B1b promoter/reporter constructs markedly reduces promoter activity after transfection. Thus, expression of the SULT2B1 isoforms is regulated, in part, by methylation of CpG dinucleotides in their proximal promoter regions and suggests an explanation for both the global silencing of SULT2B1a as well as the tissue-specific expression of SULT2B1b. Similar to human and mouse genes the rat SULT2B1 gene consists of an alternative exon I; however, as a result of exonic rearrangement, the genic locations of exons IA and IB are reversed in the rat gene. Where exon IA is located downstream of exon IB in human and mouse SULT2B1 genes, in the rat SULT2B1 gene, exon IA is located upstream of exon IB. Furthermore, unlike the case with human and mouse SULT2B1 genes where differential splicing is necessitated since a portion of exon IA is fused with exon IB to complete the SULT2B1b mRNA, this step is not required with the rat gene. Especially interesting concerning the rearrangement of the rat SULT2B1 gene is that there is not just a relocation of exon IA to be upstream of exon IB, which is the reverse of the situation in human and mouse genes, but that only that portion of exon IA encoding for the unique amino terminus of the SULT2B1a isoform is relocated. This is opportune for otherwise the SULT2B1b protein would sustain a substantial amino acid deletion rendering it inactive. The part of exon IA encoding for common amino acid sequence of the two isoforms remains in the same relative gene position as it is in the human and mouse genes and becomes exon II in the rat gene. The neurosteroid pregnenolone sulfate synthesized in glial cells plays a significant role in learning and memory performance. We investigated regulation of expression of SULT2B1a, which catalyzes the conversion of pregnenolone to its sulfoconjugate, using the rat C6 glioma cell line. C6 cells expressed SULT2B1a, whereas, neither SULT2B1b nor the prototypical steroid sulfotransferase SULT2A1 was expressed by this cell. Increasing concentration of L-glutamic acid in the presence of cyclothiazide, which prevents a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor desensitization, attenuated SULT2B1a mRNA expression; on the other hand, neither N-methyl-D-aspartate (NMDA) nor kainic acid had a significant effect. Exposure to AMPA in the presence of cyclothiazide also inhibited SULT2B1a expression. Inhibition of SULT2B1a expression by L-glutamic acid was reversed by a selective AMPA/kainate receptor antagonist and partially reversed by a specific neuronal nitric oxide synthase (NOS) inhibitor. Induction of inducible NOS by tumor necrosis factor-alpha (TNF-a_ in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression, which was partially reversed by a specific inducible NOS inhibitor. Exogenous nitric oxide (NO) donors inhibited SULT2B1a mRNA expression. Exposure to sodium nitroprusside, LPS/TNF-a, and L-glutamic acid with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, which in turn facilitates intracellular NO signaling. Cholesterol sulfate, which binds with high affinity to the retinoid-related orphan nuclear receptor (ROR)alpha, induces expression for the barrier protein, filaggrin when added to primary cultures of human keratinocytes (NHEK). Furthermore, RORalpha, SULT2B1b (cholesterol sulfotransferase) and filaggrin colocalize to the outer granular layer of the human epidermis suggesting a functional relationship. NHEK undergo terminal differentiation when subjected to an increased calcium concentration in the medium and under these conditions SULT2B1b, filaggrin and RORalpha are induced in a similar manner and time frame. Association of RORalpha with filaggrin production was demonstrated when expression of the gene for RORalpha by NHEK was inhibited by ~95% using siRNA, and this resulted in a parallel reduction in the expression of filaggrin by ~80%; furthermore, adding cholesterol sulfate to the medium failed to produce a recovery in the expression of filaggrin. Additionally, knocking down the gene for SULT2B1b also led to a reduction in filaggrin expression; however, in this case, filaggrin expression could be successfully restored following addition of cholesterol sulfate to the medium. These studies strongly suggest that cholesterol sulfate produced by the SULT2B1b activates the gene for filaggrin and does so via an interaction with RORalpha. This is the first demonstration of a molecular action for cholesterol sulfate that is reminiscent of a typical hormone. Macular degeneration, a leading cause of blindness in elderly is a complex disease that involves aging, genetics and environmental factors. Oxidized LDL (oxLDL), a component of retinal pigment epithelium (RPE) cells is cytotoxic and capable of inducing apoptosis and necrosis. Analysis of oxLDL shows predominance of 7-ketocholesterol (7kCh) and addition of this oxysterol to ARPE19 cells in free form is the cytotoxic. SULT2B1b but not SULT2B1a or SULT2A1 is expressed in retinal ARPE19 cells as well as the normal monkey retina and sulfonates 7kCh. While free 7kCh is cytooxic, the 7kCh sulfoconjugate is nontoxic.